Efficiency of Three-Calcium Phosphate Cements in Combination with Bio-Glass for Tibia Bone Recovery in Dogs; Based on Histo-Morphological Evaluations

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1 World Journal of Zoology 6 (1): 86-90, 2011 ISSN IDOSI Publications, 2011 Efficiency of Three-Calcium Phosphate Cements in Combination with Bio-Glass for Tibia Bone Recovery in Dogs; Based on Histo-Morphological Evaluations Ali Malekshahi Moghadam, Aidin Mahdavi, Davood Sharifi, Saeed Hesaraki, Mohammad Mehdi Dehghan, Abas Tavasoli and Alireza Lotfi 1 Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 2 Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 3 Department of Ceramics, Materials and Energy Research Center, Tehran, Iran 4 Department of Pathobiology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran 5 Young Researchers Club, Islamic Azad University, Shabestar Branch, Iran Abstract: The aim of this study was to investigate three-calcium phosphate (TCP) and mg contented bio-glass efficiency for healing of tibia bone defects in dogs as an animal model. Six male dogs (25-29 kg) were used. Animals were grouped; 1) dogs were treated for three months and 2) dogs were treated for six months that each group were included three dogs. Four holes (a,b,c and d) with 5mm diameter were established by dental trephine on bone body as experimental defects. Hole (a) was without any treatment (control), hole (b) were filled with TCP, hole (c) were filled with TCP (75%) and mg included bio-glass (25%) and hole (d) were filled with TCP (60%) and mg included bio-glass (40%). At day-30 and -90, samples of two groups were taken for histomorphological observations. Fibrous connective tissue and osteogenesis in woven appearance along with section of formed natural bone were histo- morphological signs of hole (a) at day-30 and -90. Also, in this sample a hole with micron area with osteogenesis and woven connective tissue were observed. While, bone with haversian system in environment of hole (b) were clear. In center of bone, a hole with micron with bone marrow cells was observed. In its environment lamellar bone with numerous haversian system were identified. In hole (c), osteogenesis in woven form with fibrous and micron area hole were observed. Also in its environment, narrow newly formed bone was seen. In hole (d), a lot of newly formed woven-like bones along with natural bone were observed. The hole was in micron area with woven bone and considerable connective tissue, without inflammatory reactions. It was concluded that three-calcium phosphate (TCP) in combination with mg contented bio-glass is so effective material for healing of tibia defects. Key words: Tibia Three-calcium phosphate Bio-glass Osteogenesis INTRODUCTION Specific synthetic materials can be a replacement for bone materials. These include natural coral, hydroxy Bone healing is one of the most critical functions of apatite, three calcium phosphate (TCP), bioactive glasses body. Occurred trauma cause soft tissues laceration, and synthetic polymers that are using in experiments on damaging and hemorrhage at bone environment that animal model as filler for bone disorders [2]. TCP is a results in blood vessels (thrombus) creation in defected compound with application in ceramic industry. It has not bone and its environment [1]. Further progress in bone structural protein and has biological adaptation without recovery occurred via callus formation. If broken bone inflammation induction in body and because of its slow pieces were locating in front to front position, bone absorption, doesn t change plasma Ca and P healing is occur quickly and without external callus concentration. TCP acts as a trellis for bone growth. Bone formation [1]. growth rate is depends on graft site, bone specify, Corresponding Author: Davood Sharifi, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran. davood.sharifi89@gmail.com. 86

2 osteoblast cells and graft fixation. Therefore when TCP is Post-Surgical Care: Antibiotics (including penicillin located in outside of periosteum, bone growth is minor. and gentamycin) were IV-injected daily. Stitches were Biological absorption of TCP occurred by passive removed at 12-day post-surgery. Clinical symptoms were solution and absorption via osteoclasts [3, 4]. Absorption daily examined to stitches removal time and after that were rate of TCP depends on chemical structure, bone density examined weekly for three months. Whole samples from and graft contacted surface. Biological absorption rate defected (experimental) segment of tibia were taken follow for TCP is considerable more than hydroxy apatite (one of dogs fatality for histo-pathological investigations. The bone cements) [3, 4]. Bio-glasses as stimulatory product samples were placed in 10% formalin buffer. in osteogenesis is without side effects and is a suitable selection for combination with TCP cement [5]. Preparation of Histo-Pathological Sections: After Optimum hollows for bone recovery experiments by TCP samples fixation and observation, tissues and fascia and are micrometer for obtaining more absorption rate muscles in bone environment, especially from upper side and better biomechanics characterizes [3, 4]. of callus were removed. After fixation, decalcification was The aim of this study was to investigate three-calcium done from bone tissue with application of 10% nitric acid. phosphate cement efficiency for healing of tibia bone Next, papering process includes dehydration, clearness, defects induced by artificial hollowing in dog as an animal dealcoholization, forming with paraffin and microtome has model, from the clinical and histo-morphological points been conducted. of view. Finally, sample sections (5-6 micrometer diameter) were taken for staining and subsequent histo-pathological MATERIALS AND METHODS studies. Six head male dogs (20-25 Kg) were randomly selected Statistical Analysis: Data obtained from observations for this experiment. Animals were kept in similar nutritional were grouped as two experimental groups; pre- and postand environmental conditions. Next, dogs were treatment, furthermore, t-test was done for detection of randomly divided into two experimental groups. TCP were significant differences between groups. obtained by calcium carbonate (Merck, Germany) and CaHPO4 (Merck, Germany) due to solid phase reaction. RESULTS TCP were mixed with bio-glass gel in three different combinations; 90%TCP: 10%Bio-glass, 75%TCP: Hole1 (control) had lower osteogenesis as compare 25%Bio-glass, 60%TCP: 40%Bio-glass. with hole2, at end of 1 month treatment period. In Prepared sample disks (5mm diameter and 3 mm microscopic observations, fibrous tissue with height) were pressed under 200 mega Pascal pressure. homogenous pink to red appearance like lacing port were Next they were heated ( C) for three hours [6]. observed in damage zone, that bone immature spicules 12 hours before surgery animals were in fasting (without were visible. In group2 spicules were mineralized and had feeding) and 4 hours without water intake. osteoid like appearance. Anesthesia was done by IV-injection of 19 mg sodium Active fibroblasts, many inflammatory mono nucleic thiopental. After anesthesia, the inside of tibia bone was macrophages and blood vessels have been observed. scraped and was washed. After one month experiment a big part of cement were not observed. The cement was surrounded with inflammatory Surgical Procedure: 10 cm in length paralleled with tibia mono nucleic macrophages. bone length were cut. Fascia and endodermal tissues In group1 hole had micron area that were separated, hereby diaphysis and metaphysis were surrounded with bone and mature tissue with minor observed. Four holes (a,b,c and d) with 5cm diameter were blood vessels and inflammatory signs were significantly established by dental trephine on bone body. Next, less. Also, signs of spicule maturation and their medullary canal were detected and observed with conversion to lamellar bone have been observed. Fatty application orthopedic drill. and active osteoblast surrounded tissue. In group1 after TCP tablets were sterilized by X-ray (25 KGY, animal keeping for three months, so many formatted for 10 hours). osteoblasts were observed. 87

3 Fig. 1: A section of 3-month-old control hole sample that a big part of defect were replaced with connective tissue and mature osteogenic blades (H&E, 200X) Fig. 3: A section of 3-month-old sample (TCP) that big part of TCP cement has been absorbed and surrounded by mature bone spicules. Formed osteogenic blades have haversian system and in overall osteogenesis and cement replacement was better than pervious condition (at the beginning of experiment). At right and button, there are so many purple granules in the cement (H&E, 284X) Fig. 2: A section of 1-month-old hole2 (TCP) that observed like reddish bulk According to Figure 1, in this group spicule extent, number and diameter was more than group1 (control). In hole2 (Figures 2 and 3), damage zone in bone was covered with netted bulk that after one month, reddish narrow osteogenic spicules and subsequently some Fig. 4: A section of 3-month-old sample (TCP: 75% and vessels have been observed. At the left and upper sides bioglass: 25%). TCP/ bioglass cement in hole of image and signs of an osteogenic spicule with active environment with micron area. The osteoblasts in its environment and in button, signs of osteogenesis was done in woven form with monocytes or macrophages can be seen. But in section of connective tissue (stickle) and natural bone (B) in this hole taken from 3-month experiment (TCP), large part near of it (H&E, 64X) of TCP has been observed and surrounded with mature spicules. Formatted osteogenic blades had haversian micron area for TCP and Mg in hole environment system and large extent, diameter and cement replacement with internal bone marrow cells in woven and better than condition observed in Figure 8. At right and Connective tissue along with compressed natural button of Figure 3, many small purple granules were bone (Figure 4). In fourth hole of one month old sample observed. In hole 3, a section were prepared from one (TCP 60% and Mg bio-glass 40%) micron month old samples (TCP 75% and Mg bio-glass) showed whole area with narrow newly-formed bone and 88

4 showed TCP and mg in whole environment with micron and osteogenesis as woven with fibroses connective tissue have been detected. A section from this bone is presented in Figure 6. DISCUSSION Fig. 5: A section of 1-month-old sample (TCP: 60% and bioglass: 40%). TCP/ bioglass cement in hole environment with micron area. The osteogenesis was done and narrow bones with connective tissue were seen, without any inflammatory signs (H&E, 64X) Fig. 6: A section of 3-month-old sample from fourth hole (TCP: 60% and bioglass: 40%). TCP/ bioglass cement in hole environment with micron area. The osteogenesis was done in woven form with fibrous connective tissue (stickle) were seen. A section of natural bone is marked with B (H&E, 64X) connective tissue in its environment have been observed. But there was not any inflammatory reaction sign (Figure 5). But, in 3-month-old samples, prepared sections from current hole (TCP 60% and Mg bioglass 40%) TCP cement is new generation bone substitutes, with potential clinical applications in orthopedics. TCP is one of the best materials for bone repair, because of their biocompatibility and osteoconductivity. The cement can be shaped into any complicated geometry or filled into any intricate cavity, within its setting period. It can adapt to the bone cavity, offering a good fixation and optimum tissue biomaterial contact necessary for stimulating bone in growth [7]. Senaha et al. [8] showed potential of bio-glasses in providing long-lasting fixation of implants to bone under weight-bearing conditions in dogs. The most notable outcome of the histological analysis is the replacement of TCP by new bone, as the healing process progresses. This is the osteoconductive property, an ideal requirement for a bone substitute which provides better strength to the healing site [9, 10] but, it seems resorption of calcium phosphate cement takes a relatively long time [11]. Mousavi et al. [12] showed that the mixture of collagen type I and calcium phosphate bone cement is a good choice for the healing of segmental bone defects and provides a more rapid regeneration of bone defects in compare with control or groups without collagen. In current experiment it was noted that TCP in combination with mg- contented bio-glass is a good situation for bone cement preparation for bone defects recovery in dog model, too (Figures 3-8). Findings of this experiment about efficiency of TCP combination with biomaterials (such as bio-glass) for acceleration of bone defect recovery is according to Mousavi et al. [12] in rabbits and Ozturk et al. [11] in rat model. Currently, Lee et al. [13] in a study on dogs reported that calcium phosphate glass cement was replaced rapidly with an abundant volume of new bone for 4 4 mm 1-wall intra-bony defects. In technical mean evidences of present study exactly were in agreement with Lee et al. [13] reports. In conclusion, three-calcium phosphate (TCP) in combination with mg contented bio-glass is so effective material for healing of tibia defects in shorter time. Also, bio-glass is a good situation for combination with TCP. 89

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